Endothelial cells line the intimal surface of all blood vessels and are the principal site within the body for synthesis of prostaglandin I2 (PGI2), a potent vasodilator and platelet antagonist. The aim of the proposed research is to identify and characterize the individual biochemical events involved in agonist-provided PGI2 synthesis in endothelial cells. Bradykinin (BK) is used as the prototypic receptor-dependent agonist, and most studies are performed with bovine pulmonary artery endothelial cells (BPAEC). The working hypothesis is that BK- induced PGI2 synthesis is initiated by an agonist-receptor interaction that is coupled to phosphoinositide (PIns) hydrolysis by a G protein. PIns-derived second messengers mobilize Ca2+ and activate phospholipase A2 (PLA2) which releases arachidonic acid (AA) for PGI2 synthesis. The involvement of G proteins and the effects of second messengers are investigated in BPAEC permeabilized with saponin. This permits agents such as GTP, nonhydrolyzable GTP analogues (GTP S), inositol-1,4,5 trisphosphate (IP3), and diacylglycerol (DAG) to be introduced into the cells to directly examine their effects on PIns hydrolysis, Ca2+ mobilization, and PLA2 activity. Correlative studies are performed in intact cells, and the time courses for the individual response parameters, eg. IP3 formation and Ca2+ mobilization, determined. Intracellular Ca2+ concentrations in BK-stimulated intact cells are measured in suspensions of BPAEC loaded with the Ca2+-sensitive fluorescent dye, Fura-2. The time course of Ca2+ changes is used as an internal time reference to distinguish biochemical events that precede and follow Ca2+ mobilization. Glucocorticoids and interleukin-1 suppress and enhance, respectively, PGI2 synthesis. Their effects on BK-induced PIns hydrolysis, Ca2+ mobilization, and PLA2 activity are compared with untreated cells in order to determine their sites of action. Purified lipocortin I and related antiphospholipase proteins are tested for their ability to mimic the effects of glucocorticoids in intact and permeabilized cells. Specific antisera to these proteins are investigated for their ability to reverse or attenuate glucocorticoid effects.